Korean J Physiol Pharmacol.  2014 Dec;18(6):475-480. 10.4196/kjpp.2014.18.6.475.

FSL-1, a Toll-like Receptor 2/6 Agonist, Induces Expression of Interleukin-1alpha in the Presence of 27-hydroxycholesterol

• Heo W
• Kim SM
• Eo SK
• Rhim BY
• Kim K

• KMID: 2285549
• DOI: http://doi.org/10.4196/kjpp.2014.18.6.475

Abstract

We investigated the question of whether cholesterol catabolite can influence expression of inflammatory cytokines via Toll-like receptors (TLR) in monocytic cells. Treatment of THP-1 monocytic cells with 27-hydroxycholesterol (27OHChol) resulted in induction of gene transcription of TLR6 and elevated level of cell surface TLR6. Addition of FSL-1, a TLR6 agonist, to 27OHChol-treated cells resulted in transcription of the IL-1alpha gene and enhanced secretion of the corresponding gene product. However, cholesterol did not affect TLR6 expression, and addition of FSL-1 to cholesterol-treated cells did not induce expression of IL-1alpha . Using pharmacological inhibitors, we investigated molecular mechanisms underlying the expression of TLR6 and IL-1alpha. Treatment with Akt inhibitor IV or U0126 resulted in significantly attenuated expression of TLR6 and IL-1alpha induced by 27OHChol and 27OHChol plus FSL-1, respectively. In addition, treatment with LY294002, SB202190, or SP600125 resulted in significantly attenuated secretion of IL-1alpha . These results indicate that 27OHChol can induce inflammation by augmentation of TLR6-mediated production of IL-1alpha in monocytic cells via multiple signaling pathways.

Keyword

27-Hydroxycholesterol; Interleukin-1; Monocytes/macrophages; TLR-6

MeSH Terms

Cholesterol
Cytokines
Inflammation
Interleukin-1
Interleukin-1alpha*
Toll-Like Receptors*
Cholesterol
Cytokines
Interleukin-1
Interleukin-1alpha
Toll-Like Receptors

Figure

• 

  Fig. 1 Expression of TLR6 in THP-1 cells in response to cholesterol and 27OHChol. (A) THP-1 cells (1×106 cells/60 mm culture dish) were serum starved in 0.1% BSA (endotoxin free) in RPMI 1640 for 24 h and treated with cholesterol (5 µg/ml) or 27OHChol (2.5 µg/ml) for 48 h. Total RNA was isolated from the cells, and TLR6 transcripts were amplified using RT-PCR. (B) THP-1 cells were serum starved in 0.1% BSA in RPMI 1640 for 24 h and treated with cholesterol (5 µg/ml) or 27OHChol (2.5 µg/ml) for 48 h. Total RNA isolated from the cells was reverse-transcribed, followed by real-time PCR in order to assess the levels of TLR6 transcripts. (C) Serum-starved THP-1 cells were treated with cholesterol or 27OHChol for 48 h, after which THP-1 cells were immunostained for cell surface TLR6. Flow cytometry was performed for analysis of fluorescence.

• 

  Fig. 2 Effects of cholesterol and 27OHChol on TLR6-mediated expression of IL-1α. (A) Serum-starved THP-1 cells were treated with cholesterol (5 µg/ml) or 27OHChol (2.5 µg/ml) for 24 h and incubated for another 24 h after addition of FSL-1 (100 ng/ml). The amount of IL-1α secreted into culture media was measured by ELISA. ***p<0.001 vs. control. (B) Serum-starved THP-1 cells were treated with cholesterol (5 µg/ml) or 27OHChol (2.5 µg/ml) for 24 h and incubated for another 24 h after addition of FSL-1 (100 ng/ml). Total RNA isolated from the cells was reverse-transcribed, followed by real-time PCR in order to assess the levels of IL-1α transcripts. ***p<0.001 vs. control.

• 

  Fig. 3 Effects of inhibitors of the PI3K/Akt pathway on TLR6-mediated expression of IL-1α. (A) Serum-starved THP-1 cells were treated with 27OHChol (2.5 µg/ml) for 24 h after pre-incubation for 2 h in the absence or presence of LY294002 or AktiIV (5 µM each). Total RNA isolated from the cells was reverse-transcribed and real-time PCR was performed for determination of the relative levels of TLR6 transcripts. ***p<0.001 vs. control; ###p<0.001 vs. 27OHChol. (B, C) Serum-starved THP-1 cells were treated with 27OHChol (2.5 µg/ml) for 24 h after pre-incubation for 2 h in the absence or presence of the indicated inhibitors (5 µM each), followed by stimulation with FSL-1 (100 ng/ml) for 24 h. (B) The amount of IL-1α secreted into culture media was measured by ELISA. ***p<0.001 vs. Control; #p<0.05 vs. 27OHChol+FSL-1; ###p<0.001 vs. 27OHChol+FSL-1. (C) Total RNA isolated from the cells was reverse-transcribed, followed by real-time PCR in order to assess the relative levels of IL-1α transcripts. ***p<0.001 vs. control; ###p<0.001 vs. 27OHChol+FSL-1.

• 

  Fig. 4 Effects of inhibitors of MAPKs on TLR6-mediated expression of IL-1α. (A) Serum-starved THP-1 cells were treated with 27 OHChol (2.5 µg/ml) for 24 h after pre-incubation for 2 h in the absence or presence of the indicated inhibitors (5 µM each). Total RNA isolated from the cells was reverse-transcribed and real-time PCR was performed for determination of the relative levels of TLR6 transcripts. ***p<0.001 vs. control; ###p<0.001 vs. 27OHChol. (B, C) Serum-starved THP-1 cells were treated with 27OHChol (2.5 µg/ml) for 24 h after pre-incubation for 2 h in the absence or presence of the indicated MAPKs inhibitors (5 µM each), followed by stimulation with FSL-1 (100 ng/ml) for 24 h. (B) The amount of IL-1α secreted into culture media was measured by ELISA. ***p<0.001 vs. Control; ##p<0.01 vs. 27OHChol+FSL-1; ###p<0.001 vs. 27OHChol+FSL-1. (C) Total RNA isolated from the cells was reverse-transcribed, followed by real-time PCR in order to assess the relative levels of IL-1α transcripts. ***p<0.001 vs. control; ###p<0.001 vs. 27OHChol+FSL-1.

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